The long-term objective of this research program is to better understand the mechanisms that couple hemostatic factors in general, and thrombin in particular, to neuroinflammatory disease (e.g., multiple sclerosis). Our central hypothesis is that thrombin and thrombin-mediated proteolysis is a critical regulator of inflammation tied to central nervous system (CNS) inflammatory disease and is mechanistically linked to the progression of inflammation and demyelination. This hypothesis is supported by strong preliminary data that suggests thrombin is integrally associated with the development of neurologic deficits associated with experimental autoimmune encephalomyelitis (EAE), an experimental setting for neuroinflammatory disease. The seminal finding that thrombin is a critical modifier of CNS inflammatory disease points to several possible fibrin (ogen)-dependent and -independent mechanisms coupling hemostatic factors to inflammation. The contribution of thrombin to CNS microglial activation and vascular leak will be established in the setting of EAE through the use of two complementary approaches: a) diminution of circulating prothrombin by use of an anti- sense oligonucleotide (ASO) gap-mer; and b) the use of a novel mouse line expressing a mutant form of prothrombin with a substrate specificity switch favoring protein C over prothrombotic substrates (Aim 1). The mechanism(s) coupling thrombin-mediated proteolysis to neuroinflammation will be further investigated by examining the role of protease activated receptor 1 (PAR-1) to the progression of paralysis associated with EAE in mice that lack PAR-1. Furthermore, the specific cell types in which PAR-signaling supports neuroinflammatory disease and loss of motor function will be determined by using a novel and only recently established mouse line carrying a floxed conditional PAR-1 knockout allele (Aim 2). These proposed studies will provide a mechanistic understanding of the cross-talk between the hemostatic and inflammatory systems in neuroinflammatory disease. These studies may also provide the fundamental foundation for novel therapeutic strategies to limit the progression of MS and other common forms of CNS disease, including Alzheimer's disease. Furthermore, the applicant will benefit from the protected, mentored time of this K08 award while pursuing studies that are an ideal platform for transitioning to independence. Through the career development plan outlined in this project, the applicant will build a record of accomplishment with a significant and novel research niche while refining the skills and tools necessary to succeed as an independent R01-funded physician-scientist.